The majority of HIV-1 infections occur at mucosal surfaces in the body. There is therefore an immediate need for potent HIV vaccines that can provide barrier protection at mucosal surfaces. While there is this need, most HIV vaccines have been developed and tested for their ability to drive systemic immune responses and not for mucosal responses. Given that systemic immunization generally does not provoke potent mucosal protection, this project will develop adenoviral gene delivery vectors targeted to specifically drive potent mucosal immune responses. This project will pursue this goal in the following Aims: Specific Aim 1. Generate New Fiber-Sigma Chimeras and Test Their Functionality In Vitro. Specific Aim 2. Test Ad-Sigma Vaccines for Their Ability to Enhance Mucosal Immunization Specific Aim 3. Determine the Role of Sigma 1 Motifs in Viral Uptake, Transduction, and Vaccination. Specific Aim 4. Test Methods to Maximize Mucosal Prime-Boost Strategies with Ad-Sigma Vaccines. This project will test both traditional replication-defective adenovirus type 5 (Ad5) vectors and Ad vectors engineered with reovirus sigma 1 protein (Ad-Sigma1) for their ability to drive mucosal immune responses against HIV-1 gag. This project will test if Ad-Sigma1 is able to enhance mucosal immunization after administration by intranasal and oral administration by virtue of its ability to target junctional adhesion molecule 1 (JAM1) and sialic acid, two receptors that are expressed on mucosal surfaces and on M cells. We will test if this mucosal-targeting vaccine targets immunologically-relevant mucosal dendritic cells, mucosal epithelial cells, and M cells to maximize mucosal vaccine efficacy and reduce side effects due to interactions with cells that do not contribute to vaccination. We will also analyze the key dependence of sigma 1 ligands and other features in mediating uptake of virions into these sites. These approaches will be tested in humanized HLA-A*0201 transgenic mice to give guidance for future human tests. Successful pursuit of this project will enable more specific and less dangerous adenoviral vaccines. This project will provide vaccine efficacy and safety data for these three Ad vaccine approaches in mice as a precursor to testing in rhesus macaques and in humans.